Physical properties of transmembrane proteins, including lateral mobility, rotational mobility, steric interactions, and high and low affinity binding interactions, are important determinants of cell membrane structure and function. Erythroid cells provide an ideal system for study of the molecular mechanisms that regulate such physical properties. Recent studies in human red blood cells have led to a working model of the molecular mechanisms controlling the average lateral and rotational mobility of band 3, the red cell anion exchanger. Steric hindrance interactions provided by the spectrin based membrane skeleton appear to regulate the rate of band 3 lateral diffusion; low affinity binding interactions with the membrane skeletal proteins ankyrin and protein 4.2 to regulate the rate of band 3 rotational diffusion; high affinity binding interactions with ankyrin to determine the fraction of rotationally immobile band 3; and band 3 oligomerization to control the fraction of laterally immobile band 3. This model is tested in the present proposal, at the level of individual band 3 molecules, in three related erythroid cell systems: mature red cells from patients with hereditary hemolytic anemias and defects in spectrin, ankyrin, band 3, protein 4.1, and protein 4.2; mature red cells from mouse strains with hereditary hemolytic anemias and defects in spectrin and ankyrin, and from transgenic mouse strains with engineered defects in ankyrin, band 3, and protein 4.2; and differentiating human and murine erythroid cells in culture. The physical properties of the major RBC sialoglycoprotein, glycophorin A, are also examined in mature red cells and in differentiating erythroid cells. Single particle tracking is used to distinguish among four modes of translational motion of individual protein molecules, including unrestricted diffusion, constrained diffusion, directed movement, and immobilization. Laser optical tweezers are employed to quantify the strength of high and low affinity binding interactions involving individual protein molecules in the native membrane environment. Fluorescence photobleaching recovery and polarized fluorescence depletion are used to measure the average lateral and rotational mobility, respectively, of transmembrane proteins. Membrane biochemistry and ultrastructure are correlated with transmembrane protein physical properties at the level of individual molecules. These studies will lead, for the first time, to a molecular understanding of the physical forces constraining transmembrane protein mobility. Elucidation of the nature and magnitude of such forces will help to explain the pathophysiology of membrane disorders including hereditary spherocytosis and hereditary elliptocytosis.